EP1178822B1 - Vaccin anticancereux a cellules entieres comprenant des cellules malignes et non-malignes co-cultivees - Google Patents

Vaccin anticancereux a cellules entieres comprenant des cellules malignes et non-malignes co-cultivees Download PDF

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EP1178822B1
EP1178822B1 EP00931400A EP00931400A EP1178822B1 EP 1178822 B1 EP1178822 B1 EP 1178822B1 EP 00931400 A EP00931400 A EP 00931400A EP 00931400 A EP00931400 A EP 00931400A EP 1178822 B1 EP1178822 B1 EP 1178822B1
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cells
cultured
malignant
culture
cell
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EP1178822A2 (fr
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Peter Onyvax Limited Smith
Darren Onyvax Limited STEVENSON
Haj Onyvax Limited CHANA
Peter Onyvax Limited THRAVES
Andrew Sutton
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Onyvax Ltd
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Priority claimed from GB0008032A external-priority patent/GB0008032D0/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5152—Tumor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1352—Mesenchymal stem cells
    • C12N2502/1358—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/28—Vascular endothelial cells

Definitions

  • New vaccines for use as immunotherapeutic agents in the treatment of cancer are described.
  • First generation whole cell vaccines relied on traditional methodologies in the culture and production of the autologous or allogeneic cell based vaccines, whereby autologous tumour cells or immortalised allogeneic tumour cell lines were cultured in static two dimensional (2D) culture conditions.
  • This invention relates to a co-culture techniques which leads to significant enhancement of the phenotype of cell lines towards a more in vivo phenotype resulting in a vaccine reflecting a more realistic phenotypic representation of an in vivo tumour.
  • We describe alternative culture techniques which offer significant scale up and processing benefits and in addition significantly improve the activity of the whole-cell vaccine.
  • vaccines produced by the various co-culture techniques described have additional advantages over single cell suspension vaccines in the mode of presentation to the immune system which leads to enhanced performance in treating the disease.
  • cancerous cells contain numerous mutations, qualitative and quantitative, spatial and temporal, relative to their non-malignant, non-cancerous counterparts and that at certain periods during tumour growth and spread a proportion of the cancerous cells are capable of being recognised by the host's immune system as abnormal.
  • Cancer cells express certain tumour-specific antigens (TSA) and tumour-associated antigens (TAA) that may serve as markers or targets for the immune system.
  • TSA tumour-specific antigens
  • TAA tumour-associated antigens
  • Tumours have the remarkable ability to counteract the immune system in a variety of ways which may include: down-regulation of the expression of potential target proteins; mutation of potential target proteins; down-regulation of surface expression of receptors and other proteins; down-regulation of MHC class I and II expression thereby disallowing direct presentation of TAA or TSA peptides; down-regulation of co-stimulatory molecules leading to incomplete stimulation of T-cells leading to anergy; shedding of selective, non representative membrane portions to act as decoy to the immune system; shedding of selective membrane portions to anergise the immune system; secretion of inhibitory molecules; induction of T-cell death; and many other ways.
  • Cell-based vaccines fall into two categories. The first, based on autologous cells, involves the removal of a biopsy from a patient, cultivating tumour cells in vitro , modifying the cells through transfection and/or other means, irradiating the cells to render them replication-incompetent and then injecting the cells back into the same patient as a vaccine. Although this approach enjoyed considerable attention over the past decade, it has been increasingly apparent that this individually-tailored therapy is inherently impractical for several reasons.
  • tumour biopsy used to prepare the autologous vaccine will have certain growth characteristics, interactions and communication with surrounding tissue that makes it somewhat unique.
  • the second type of cell-based vaccine describes the use of allogeneic cells which are be genetically (and hence immunologically) mismatched to the patients. Allogeneic cells benefit from the same advantages of multivalency as autologous cells. In addition, as allogeneic cell vaccines can be based on immortalised cell lines which can be cultivated indefinitely in vitro, this approach does not suffer the lead-time and cost disadvantages of autologous approaches. Similarly the allogeneic approach offers the opportunity to use combinations of cells types which may match or anticipate the disease profile of an individual in terms of stage of the disease, the location of the lesion and potential resistance to other therapies.
  • 2D culture - cells grown in static plastic flasks within incubators, with or without CO 2 atmospheric supplementation
  • the cells grow in two dimensions only as a flat monolayer or sheet of cells adherent to the plastic or glass vessel. Cell growth usually becomes contact inhibited at confluence and attempts to grow the cells beyond this point usually results in the loss of the culture.
  • the growth of cells in 2D culture relies on the serial passage of cells, wherein the cells are repeatedly removed from the substratum and subjected to a harsh cycle of centrifugation, washing and manipulation in suspension.
  • the cells are grown and then harvested by mechanical means or treatment with trypsin to remove the cells from the substratum.
  • trypsin other proteolytic enzymes or other agents used for the purpose is to break attachment of the cells to the flask substrate.
  • Proteolytic treatment inevitably removes some protein molecules from the surface of the cells, which may result in loss of potential epitopes that enable the recipient to raise an humoral or cell based immune response to the tumour.
  • proteolysis may remove MHC-I molecules that could present peptides in a direct manner to patient T cells, or adhesion molecules that offer co-stimulatory activity to direct presentation.
  • proteolysis may simply remove aberrantly expressed TAA or TSA which when processed through a cross-priming or other route could also contribute to an anti-tumour response.
  • TAA or TSA which when processed through a cross-priming or other route could also contribute to an anti-tumour response.
  • proteolysis may simply remove aberrantly expressed TAA or TSA which when processed through a cross-priming or other route could also contribute to an anti-tumour response.
  • there is relatively little opportunity to manipulate the cell phenotype since, in order to attain the quantity of cells required, a number of passages under standard growth conditions are required and attempts to drive the cells into a stressed or apoptotic or necrotic or otherwise altered phenotype prior to harvest results in significant losses of fragile cells at harvest undermining the feasibility of producing vaccine at a practical scale.
  • Hitherto described cell based cancer vaccines are based on i) populations of primary cultures of autologous tumour cells; ii) single populations of allogeneic immortalised tumour cell lines; iii) allogeneic immortalised cell lines as many as three or four in combination and iv) genetically modified fibroblasts in combination with tumour cells.
  • WO/99 19462. discloses a whole cell cancer vaccine wherein the malignant cells are cultured with other non-malignant cells. This results in a vaccine in which the non-malignant cells have an enhanced cytokine secretion and co-stimulatory molecules expression, which renders the whole cell vaccine more effective.
  • the present invention describes culture methods for large scale production of vaccines; phenotype adjustment of established cell lines; co-culture techniques; specific compositions of immortalised tumour cell lines and immortalised non-malignant cells; compositions comprising immortalised tumour cells and autologous non-malignant cells; processing methods for the production of the vaccines; analytical techniques to evaluate phenotype adjustment and new compositions which display increased immunogenicity in standard models of tumour prevention and therapy.
  • this invention describes a new strategy, targeting the immune response not only to the tumour itself but also to the local tumour environment and the non-malignant cells which comprise that environment.
  • tumour cells are intensely immunosuppressive where all facets of immune surveillance are inhibited, from inhibition of T cell and antigen presenting cell (APC) migration, to inhibition of APC maturation and induction of T cell anergy.
  • APC antigen presenting cell
  • Many of the immunosuppressive activities found in the immediate local tumour environment are mediated by soluble factors released by the tumour cells, such as IL-10 and TGF ⁇ .
  • soluble factors released by the tumour cells such as IL-10 and TGF ⁇ .
  • Tumour-adjacent cells are induced to secrete or express enhanced levels of receptors or other proteins (e.g.
  • tumour-adjacent non-malignant cells are aberrant to distant non-malignant cells in that they are induced to express proteins that distant counterparts do not; this will be reflected in a qualitatively and/or quantitatively modified spectrum of peptides displayed on the tumour-adjacent cells MHC-I molecules which can be recognised by the immune system as aberrant.
  • this embodiment targets the local tumour micro-environment in addition to the tumour itself.
  • This embodiment describes the preparation of a mixed cell type vaccine, in which tumour cell lines are grown in intimate contact with a non-malignant component.
  • the growth of this mixed cell type vaccine can be achieved by a number of means, for example co-culture in 2D flasks, growth in transwell inserts where conditioned medium exerts its effect on the tumour line or growth in a 3D mixed culture system.
  • There are significant benefits to a mixed culture approach in that the literature reports that in many cases celi-ceil contact is required to elicit reciprocal phenotypic changes in both tumour and non-tumour components (e.g. Fibroblast mediated differentiation in human breast carcinoma cells (MCF-7) grown as nodules in vitro , Int. J.
  • MCF-7 human breast carcinoma cells
  • This invention describes the use of 3D microgravity culture to grow significant quantities of tumour cells and co-cultured cells for use as vaccines.
  • a 500 ml 3D microgravity vessel we can achieve the same vaccine production of mammalian tumour cell lines as we could obtain by culturing 300 T175 flasks or 30 1,700 cm 2 roller bottles.
  • a further embodiment of this invention is the use of 3D co-culture to achieve a mixed cell type vaccine comprising an immortalised tumour cell line in combination with an immortalised or non-immortalised non-malignant cell line such as epithelial cells, stromal fibroblasts, bone stromal cells, endothelial cells, palisade cells or any other immortalised or non immortalised non-malignant cell type.
  • an immortalised or non-immortalised non-malignant cell line such as epithelial cells, stromal fibroblasts, bone stromal cells, endothelial cells, palisade cells or any other immortalised or non immortalised non-malignant cell type.
  • an immortalised or non-immortalised non-malignant cell line such as epithelial cells, stromal fibroblasts, bone stromal cells, endothelial cells, palisade cells or any other immortalised or non immortalised non-malignant cell type.
  • an immortalised or non-immortalised non-malignant cell line
  • Two immortalised neuroendocrine tumour lines are known in the literature, the prostatic derived lung metastatic neuroendocrine line CRL5813 (NCI-H660) and the colorectal neuroendocrine line COLO320DMF, both of which secrete neuroendocrine factors such as parathyroid hormone, bombesin, etc.
  • the cultures may be co-seeded with two or more cell types taken from independent 2D culture flasks, or the culture may be sequentially seeded for example with a stromal fibroblast first, followed some one or two days later by a tumour cell line.
  • a further aspect of this invention is a direct process of formulation of cells harvested from 3D microgravity culture without the need to treat the cells with trypsin. This process therefore has the advantage of significantly increasing the yield of cells following harvest, whereas for traditionally grown 2D cultures significant losses are incurred during the trypsinization, neutralization, washing and formulation. Cells harvested as spheroids tend to be significantly more robust to handle and wash, thereby leading to enhanced recoveries.
  • a further embodiment of this invention is the use of mixed 3D microgravity culture incorporating non-malignant cell lines to enhance the phenotype of cell lines which hitherto have not been grown in mixed 3D culture.
  • a further embodiment of this invention is enhanced immunogenicity arising from the use of non-malignant cells in combination with tumour cell lines arising from direct presentation of peptides on the MHC-I molecules of the non-malignant cell line, particularly if the non-malignant cell line is matched or partially matched to the recipient.
  • the peptides may be released from the tumour cell lines during growth, processing and irradiation of the 3D cell mass, or may be released during initial interaction of macrophages and dendritic cells at the time of injection.
  • a separate embodiment of this invention is the use of peptide extracts and other sub-cellular fractions derived from cells and co-cultures of cells grown under non-standard conditions. This includes cells grown on roller bottles, on microcarriers, on polyester fibre discs, 3D culture on non adherent flasks and 3D culture under microgravity conditions.
  • the sub-cellular fractions or peptide fractions derived from the cells offer the opportunity to significantly increase the active component of a given vaccine.
  • This aspect of the invention also allows for further fractionation of the sub-cellular fractions or peptides to select the active principles and to simplify the vaccine composition if so required.
  • a further embodiment of this invention is the production, composition and use of a mixed autologous / allogeneic cell vaccine comprising autologous non-malignant cells taken from an intended recipient of the vaccine grown in culture with an allogeneic immortalised tumour cell line.
  • the autologous non-malignant cells have non-malignant levels of MHC-I and will thus have the capability of presenting in the context of autologous MHC-I peptides released from lysed, destroyed or apoptotic tumour cells with which they have been grown.
  • the murine melanoma cell line K1735 was grown with the murine fibroblast cell line 3T3 at a ratio of 1:1.
  • the 3T3 cells were seeded at 5 x 10 6 cells in a 50 ml cassette for a microgravity instrument (Synthecon) and allowed to grow for 2 days in DMEM 2% foetal calf serum, 10 mM glutamine.
  • a further inoculum of 5x10 6 cells of either K1735 was made into the 50 ml rotating cassette (Synthecon) and the culture inoculated for a further 6-9 days.
  • the resulting spheroids were harvested and washed by light centrifugation in DMEM at day 8-11 and taken for further analysis.
  • the human prostatic epithelial tumour line NIH1542 was grown with the non-malignant human prostatic epithelial line PNT2-C2 at a 1:1 ratio in a 3D microgravity culture vessel (Synthecon).
  • the cell lines were co-inoculated at 5x10 6 cells each and cultured in KSFM for a period of 6-11 days until a significant biomass of spheroids had accumulated.
  • the cell mass was harvested and washed in serum free medium and then trypsinized to prepare the sample for analysis.
  • the human prostatic epithelial tumour line NIH1542 was grown with the human prostatic neuroendocrine epithelial line CRL-5813 at a 1:1 ratio in a 3D microgravity culture vessel (Synthecon).
  • the cell lines were co-inoculated at 5x10 6 cells each and cultured in KSFM for a period of 6-11 days until a significant biomass of spheroids had accumulated. ).
  • two separate 2D cultures were seeded and grown to near confluence.
  • the cells were trypsinized and the mixture of cells from 2D and 3D co-culture were analyzed for the level of apoptosis by annexin and propidium iodide staining and. also for phenotypic expression of various markers.
  • the murine melanoma cell line K1735 was grown with the murine fibroblast cell line 3T3 at a ratio of 1:1.
  • the 3T3 cells were seeded at 5 x 10 6 cells in a 50 ml cassette for a microgravity instrument (Synthecon) and allowed to grow for 2 days in DMEM 2% foetal calf serum, 10 mM glutamine.
  • a further inoculum of 5x10 6 cells of either K1735 was made into the 50 ml rotating cassette (Synthecon) and the culture inoculated for a further 6-9 days.
  • the cells were allowed to grow for a period of 7-14 days with intermediate media and growth factor replenishment.
  • the cassette was emptied into a centrifuge vessel and lightly centrifuged to pellet the biomass.
  • the visible and invisible spheroids were washed three times in PBS or other inert media, irradiated at 50-300 Gy and then either used directly in the protection experiment or; formulated in appropriate freezing mixture (10% DMSO in Hanks) and aliquoted on the basis of either cell number determined by trypsinization and counting or on a protein or nucleic acid basis. No trypsinization was required to remove the vaccine cells from the culture vessel thereby retaining valuable TAA and TSA which will elicit an anti-tumour response in the recipient mammal.
  • the spheroids are significantly easier to harvest as a result of their size, requiring less aggressive centrifugation than is normally required for individual cells which leads to significant enhancement of the recovered cell mass.
  • mice Groups of 8 C57/B6 mice were immunized with 1-10 x10 6 K1735 / 3T3 spheroids and compared to 2D flask grown K1735 cells irradiated at 150 Gy, at T-10 days. The mice were then challenged on Day 0 with 5x10 5 live B16F10 cells and then monitored for tumour growth.
  • Cell lines ONYCAP-1, ONYCAP-23, P4E6 and SHMAC-4 were seeded at 1 x 10 6 cells onto confluent monolayers of NHBMSC cells in T175 culture flasks. Co-cultures were maintained for 2 days using keratinocyte growth medium supplemented with 5% foetal calf serum. Cells were harvested by scraping from the surface of the plastic and washed in Hanks balanced salt solution before RNA extraction.
  • Onycap1 and Onycap 23 have been deposited under the Budapest Treaty at ECACC on 28 March 2000 under the Accession numbers 00032802 and 00032801 respectively.
  • a double extraction was performed using TRI REAGENT (Sigma #T9424).
  • the reagent was added directly to the washed cell pellets and samples were allowed to stand for 5 minutes before the addition of chloroform. Samples were vortexed and allowed to stand for a further 10 minutes at room temperature then centrifuged at 12,000xg for 15 minutes.
  • RNA precipitated with isopropanol RNA pellets were washed with 75% ethanol, dried and re-suspended in TE buffer.
  • RNA sample was treated with Deoxyribonuclease I (Life Technologies #18068-015) to ensure there was no contamination with genomic DNA. Reactions were incubated for 15 minutes at room temperature then the DNase inactivated by the addition of 25mM EDTA and heating to 65°C for 10 minutes.
  • Reverse transcription was performed using the 1st strand cDNA synthesis kit for RT-PCR (AMV) from Boehringer Mannheim (#1 483 188). Reactions were incubated at 25°C for 10 minutes then at 42°C for 1 hour. The AMV enzyme was denatured by heating to 99°C for 5 minutes then the reaction was cooled to 4°C.
  • AMV 1st strand cDNA synthesis kit for RT-PCR
  • PCR primers were designed from published sequences. Verification of cDNA integrity was achieved by PCR amplification using GapdH housekeeping primers. Amplification reactions with the external primer set were carried out using thermocycler program (1). The internal nested PCR set was run on program (2) below.
  • the PCR samples were run in pairs.
  • the prostate cells alone then the same cells grown in co-culture with NHBMSC followed by NHBMSC alone then a positive and negative control Figure 1.
  • PSA expression was not detected in 3 out of 4 prostate cells grown alone however, cDNA isolated from all of the cell lines grown in co-culture with NHBMSC produced a band at 200bp following 2 rounds of PCR amplification suggesting an up-regulation of PSA as a result of co-culture with bone marrow stromal cells.
  • Cell line ONYCAP-23 were seeded at 1 x 10 6 cells onto confluent monolayers of NHBMSC cells in T175 culture flasks. Co-cultures were maintained for 2 days using keratinocyte growth medium supplemented with 5% foetal calf serum. Cells were harvested by scraping from the surface of the plastic and washed in Hanks balanced salt solution before RNA extraction.
  • a double extraction was performed using TRI REAGENT (Sigma #T9424).
  • the reagent was added directly to the washed cell pellets and samples were allowed to stand for 5 minutes before the addition of chloroform. Samples were vortexed and allowed to stand for a further 10 minutes at room temperature then centrifuged at 12,000xg for 15 minutes.
  • RNA precipitated with isopropanol RNA pellets were washed with 75% ethanol, dried and re-suspended in TE buffer.
  • RNA sample was treated with Deoxyribonuclease I (Life Technologies #18068-015) to ensure there was no contamination with genomic DNA. Reactions were incubated for 15 minutes at room temperature then the DNase inactivated by the addition of 25mM EDTA and heating to 65°C for 10 minutes.
  • Reverse transcription was performed using the 1st strand cDNA synthesis kit for RT-PCR (AMV) from Boehringer Mannheim (#1 483 188). Reactions were incubated at 25°C for 10 minutes then at 42°C for 1 hour. The AMV enzyme was denatured by heating to 99°C for 5 minutes then the reaction was cooled to 4°C.
  • AMV 1st strand cDNA synthesis kit for RT-PCR
  • PSA PCR primers were designed from published sequences tabulated below. Verification of cDNA integrity was achieved by PCR amplification using GapdH housekeeping primers. Amplification reactions with the external primer set were carried out using thermocycler program (1). The internal nested PCR set was run on program (2) below.
  • thermocycler program was given below and were run on the thermocylcer
  • PCR samples were run in pairs on agarose gels.
  • tumour elicited stroma shows aberrant expression of proteins in response to the tumour cells.
  • K1735 (P25) cells were grown in RPMI 1640 medium supplemented with 10% FCS and 2 mM L-glutamine, trypsinised, irradiated at 100 Gy and formulated into 10%DMSO in RPMI at a cell density of 5x106 cells / ml prior to cryopreservation at -1800C.
  • a K1735 tumour grown in a C3H mouse was excised and chopped into small pieces before being forced through a 70um gauze, irradiated at 100 Gy and formulated in 10%DMSO in RPMI at a cell density of 5x106 cells / ml, then cryopreserved at -1800C.
  • B16.F10 mouse melanoma clone G10 was grown in RPMI 1640 medium supplemented with 10% FCS and 2 mM L-glutamine, trypsinised and used to inoculate groups of 5 C57BL6 mice at a viable dose of 1x104 cells in 200ul of medium. Three days following viable tumour challenge the mice were dosed with 1x106 of either in vitro grown K1735 or in vivo grown tumour or PBS injected mice and subsequently on three other occasions on days 6 10 and 13 post tumour challenge. Tumour volume and survival were measured (sacrifice when tumour attained 15x15mm) at three day intervals starting at day 11 post tumour challenge.
  • Cell line DU145 was seeded at 1 x 10 6 cells onto confluent monolayers of normal human prostatic stromal fibroblasts (NHPSF) in T175 culture flasks. Co-cultures were maintained for 2 days using in MEM medium supplemented with 10% FCS and 2 mM L-glutamine. Cells were harvested by scraping from the surface of the plastic and washed in Hanks balanced salt solution before RNA extraction.
  • NHSF normal human prostatic stromal fibroblasts
  • Normal human prostatic stromal fibroblasts were grown to confluency in a T175 flask in MEM medium supplemented with 10% FCS and 2 mM L-glutamine.
  • Cell line DU145 was seeded at 1 x 10 6 cells into T175 flasks and grown in MEM medium supplemented with 10% FCS and 2 mM L-glutamine and grown to confluency. Cells were harvested by scraping from the surface of the plastic and washed in Hanks balanced salt solution before RNA extraction.
  • a double extraction was performed using TRI REAGENT (Sigma #T9424).
  • the reagent was added directly to the washed cell pellets and samples were allowed to stand for 5 minutes before the addition of chloroform. Samples were vortexed and allowed to stand for a further 10 minutes at room temperature then centrifuged at 12,000xg for 15 minutes.
  • RNA precipitated with isopropanol RNA pellets were washed with 75% ethanol, dried and re-suspended in TE buffer.
  • RNA sample was treated with Deoxyribonuclease I (Life Technologies #18068-015) to ensure there was no contamination with genomic DNA. Reactions were incubated for 15 minutes at room temperature then the DNase inactivated by the addition of 25mM EDTA and heating to 65°C for 10 minutes.
  • Labelled cDNA was used to probe a Clontech AtlasTM array (human cancer array II) following the manufacturers protocol for hybridisation and washing. Images were recorded utilising a phosphor imager and recorded files analysed with the Clontech Atlas ImageTM software.
  • tumour cell lines which are up or down regulated by growth of tumour cell lines on stromal cells from various organ compartments.
  • tumour cell lines with varying degrees of invasivity or metastatic capability in combination with stromal cells such as prostatic fibroblasts, bone stromal fibroblasts, or endothelial cells it is possible to identify significant genes which are temporally and spatially expressed as a result of the intimate contact between tumour and stromal cell and thus enable the identification of antigens which are thus meaningful and viable potential targets for immunotherapy or other therapeutic strategies.
  • Cell line DU145 was seeded at 1 x 10 6 cells onto confluent monolayers of normal human bone stromal fibroblasts (NHBSF) in T175 culture flasks. Co-cultures were maintained for 2 days using in MEM medium supplemented with 10% FCS and 2 mM L-glutamine. Cells were harvested by scraping from the surface of the plastic and washed in Hanks balanced salt solution before RNA extraction.
  • NHSF normal human bone stromal fibroblasts
  • Normal human bone stromal fibroblasts were grown to confluency in a T175 flask in MEM medium supplemented with 10% FCS and 2 mM L-glutamine. Cells were harvested by scraping from the surface of the plastic and washed in Hanks balanced salt solution before RNA extraction.
  • Cell line DU145 was seeded at 1 x 10 6 cells into T175 flasks and grown in MEM medium supplemented with 10% FCS and 2 mM L-glutamine and grown to confluency. Cells were harvested by scraping from the surface of the plastic and washed in Hanks balanced salt solution before RNA extraction.
  • a double extraction was performed using TRI REAGENT (Sigma #T9424).
  • the reagent was added directly to the washed cell pellets and samples were allowed to stand for 5 minutes before the addition of chloroform. Samples were vortexed and allowed to stand for a further 10 minutes at room temperature then centrifuged at 12,000xg for 15 minutes.
  • RNA precipitated with isopropanol RNA pellets were washed with 75% ethanol, dried and re-suspended in TE buffer.
  • RNA sample was treated with Deoxyribonuclease I (Life Technologies #18068-015) to ensure there was no contamination with genomic DNA. Reactions were incubated for 15 minutes at room temperature then the DNase inactivated by the addition of 25mM EDTA and heating to 65°C for 10 minutes.
  • Reverse transcription was performed using the 1st strand cDNA synthesis kit for RT-PCR (AMV) from Boehringer Mannheim (#1 483 188). Reactions were incubated at 25°C for 10 minutes then at 42°C for 1 hour. The AMV enzyme was denatured by heating to 99°C for 5 minutes then the reaction was cooled to 4°C. 32P or 33P deoxynucleotides were used to label the cDNA for subsequent probing of the arrays. RNA from DU145 and NHBSF cultures were mixed in equal proportions and the same quantity of RNA used in the reverse transcription as was used for the co-culture RNA.
  • AMV 1st strand cDNA synthesis kit for RT-PCR
  • Labelled cDNA was used to probe a Clontech AtlasTM array (human array II) following the manufacturers protocol for hybridisation and washing. Images were recorded utilising a phosphor imager and recorded files analysed with the Clontech Atlas ImageTM software.
  • tumour cell lines which are up or down regulated by growth of tumour cell lines on stromal cells from various organ compartments.
  • tumour cell lines with varying degrees of invasivity or metastatic capability in combination with stromal cells such as prostatic fibroblasts, bone stromal fibroblasts, or endothelial cells it is possible to identify significant genes which are temporally and spatially expressed as a result of the intimate contact between tumour and stromal cell and thus enable the identification of antigens which are thus meaningful and viable potential targets for immunotherapy or other therapeutic strategies.
  • Results are shown for patient numbers 1, 2, 3, and 4 who had received a series of vaccinations with human prostate cell lines, PNT-2, NIH1542, DU145 and LnCap in a clinical trial to evaluate the safety and immunogenicity of these cell lines.
  • Results are given for cell lysates namely, NIH1542, LnCap, DU-145, PNT-2 and also a co-culture of DU145 and NHBSF celis in such a way that the same cell number equivalent is used in each proliferation assay.
  • the co-culture lysate has led to a significant improvement in CD4 and CD8 proliferative response in 3 out of 4 patients. This is indicative of a more representative spectrum of antigens being produced in co-culture than in the mono-culture of the DU-145 alone.
  • the rat PA3 prostate tumour line is a relatively weak allogeneic immunogen in the copenhagen rat challenged with autologous MatLyLu prostate cells.
  • PA3 rat prostate tumour cells grown in T185 flasks and PA3 cells co-cultured with bone stromal fibroblasts and YPEN endothelial cells in T185 flasks as a vaccine in a protection model.
  • PA3 Clone 1 cells (1 x 10 6 ) cells were seeded into a T175 tissue culture flask in 35 ml of RPMI 1640 culture medium supplemented with 10% FCS/2 mM Glutamine. Cells were allowed to grow until confluent under conditions of 37°C/5% CO 2 . Confluent cells were harvested by washing the monolayer with 10ml Hank's buffered salt solution, followed by 5ml trypsin/EDTA solution to remove cells from the flask. Once all cells had lifted away from the flask surface the trypsin reaction was stopped by the addition of 5ml FCS. The 10ml of cell suspension was collected in a 50ml tube, centrifuged at 18,000 rpm for 3 minutes. The resultant pellet was re-suspended into 10ml PBS and stored on ice, prior to irradiation
  • PA3 Clone 1 cells (1 x 10 5 ) and YPEN cells (1 x 10 5 ) were seed simultaneously into a T75 tissue culture flask containing 15ml RPMI 1640 culture medium supplemented with 10% FCS/2mM Glutamine. Cells were allowed to grow until confluent and harvested by cell scraping, pelleted by centrifugation at 18,000 rpm for 3 min, and re-suspended in 10ml PBS. Cells were stored on ice prior to irradiation.
  • PA3 Clone 1 cells (1 x 10 5 ), YPEN cells 1 x 10 5 and RBMS (1 x 10 5 ) were seeded simultaneously into a T75 tissue culture flask containing 15ml RPMI 1640 culture medium supplemented with 10% FCS/2mM Glutamine. Cells were allowed to grow until confluent and harvested by cell scraping, pelleted by centrifugation at 18,000 rpm for 3 min, and re-suspended in 10ml PBS. Cells were stored on ice prior to irradiation.
  • PA3 Clone 1 cells (1 x 105) and (1 x 105) RBMS were seeded simultaneouslyinto a T75 tissue culture flask containing 15ml RPMI 1640 culture medium supplemented with 10% FCS/2mM Glutamine. Cells were allowed to growuntil confluent and harvested by cell scraping, pelleted by centrifugation at 18,000 rpm for 3 min, and re-suspended in 10ml PBS. Cells were stored once prior to irradiation.
  • Tubes containing cell suspensions were irradiated with 6 x 25 Gy (Total: 150 Gy). A cell count is then performed on each sample using a haemocytometer. Briefly 10 ⁇ l of each cell suspension in mixed with 90 ⁇ l of tryphan blue and approx. 10 ⁇ l was loaded under the coverslip of the haemocytometer. A cell count was performed using a x20 objective lens. Each cell suspension was then centrifuged at 18,000 rpm for 3 minutes. The resultant pellet was re-suspended into vaccine freezing medium (RPMI 1640/8%FCS/8%DMSO) at 5 x 10 6 cells per ml of freezing medium. Suspensions were then aliquoted into cryovials at 1.2ml per vial; vials were then placed into a -80°C freezer overnight prior to storage in liquid nitrogen.
  • vaccine freezing medium RPMI 1640/8%FCS/8%DMSO
  • the challenge material used for these experiments is the cell line Mat-Ly-Lu (Wild Type)
  • Mat-Ly-Lu cells (1 x 10 6 ) were seeded into a T175 tissue culture flask in 35 ml of RPMI 1640 culture medium supplemented with 10% FCS/2 mM Glutamine. Cells were allowed to grow until confluent under conditions of 37°C/5% CO 2 . Confluent cells were harvested by washing the monolayer with 10ml Hank's buffered salt solution, followed by 5ml trypsin/EDTA solution to remove cells from the flask. Once all cells had lifted away from the flask surface the trypsin reaction was stopped by the addition of 5ml FCS. The 10ml of cell suspension was collected in a 50ml tube, centrifuged at 18,000 rpm for 3 minutes.
  • the resultant pellet was re-suspended into 10ml PBS and stored on ice.
  • a cell count was performed as detailed above and cell suspension adjusted to 5 x 10 5 per ml and aliquoted into cryovials at 1.2 ml per vial. Vials were then held on ice prior to injection.
  • Irradiated Mat-Ly-Lu cells (0.2 ml) were injected into shaved left hind flank of a copenhagen rat. 1 vial is sufficient to inject 5 animals (1 experimental group). See below for injection regime for protection and therapy
  • Tumor dimensions were collected using calipers, two measurements-longitudinal and transverse were taken for each tumor. These values were multiplied together to give an indication of tumor size. A kill size of 25 mm was set such that any tumor reaching size in one of the two measurable dimension was culled by exposure to a rising concentration of CO 2 and cervical dislocation. Kill curves were then generated.
  • the rat PA3 prostate tumour line is a relatively weak allogeneic immunogen in the copenhagen rat challenged with autologous MatLyLu prostate cells.
  • PA3 rat prostate tumour cells grown in 3D microgravity culture and PA3 cells co-cultured with bone stromal fibroblasts and YPEN endothelial cells in 3D microgravity culture as a vaccine in rat therapy and protection models.
  • the roller cassette were filled with 35 ml culture medium (RPMI 1640 10% FCS/2mM Glutamine) through the screw cap, to this 6 x 10 6 cells suspended 6 ml of culture medium was added. The roller was then topped up to full (approx. 50ml) with -10 ml culture medium and the screw cap replaced. A 20ml syringe fitted with a 20 ⁇ m filter attached was screwed onto the outlet valve of the roller cassette. A second 20ml syringe fitted with a 20 ⁇ m was filled with culture medium and screwed onto the inlet valve of the roller cassette. Both valves on the cassette were opened and pressure applied to the inlet syringe to force any remaining air bubbles from within the cassette.
  • 35 ml culture medium RPMI 1640 10% FCS/2mM Glutamine
  • valves were closed and any remaining culture medium was aspirated off and the valves sealed with stoppers.
  • the roller cassettes were attached to the roller apparatus and rotated at 16 rpm at 37°C/5% CO 2 . Cultures were allowed to continue for 3-4 days when they were harvested by unscrewing the cap on the roller and pouring the contents into a 50ml tube. The cells were washed three times by centrifugation and resuspension and stored on ice, prior to irradiation.
  • the same growth methodology was used to produce co-cultures of PA3/YPEN, PA3/RBMS and PA3/YPEN/RBMS in 3D microgravity.
  • the total seed into the 3D Cassette was retained at 6x10 6 cells (3x10 6 cells/cell type for two cell types or 2x10 6 cells /cell type for three cell types).
  • the growth and harvest of the co-cultures is as described in the previous section.
  • Tubes containing cell suspensions were irradiated with 6 x 25 Gy (Total: 150 Gy). A cell count is then performed on each sample using a haemocytometer. Briefly 10 ⁇ l of each cell suspension is mixed with 90 ⁇ l of tryphan blue and approx. 10 ⁇ l was loaded under the coverslip of the haemocytometer. A cell count was performed using a x20 objective lens. Each cell suspension was then centrifuged at 18,000 rpm for 3 minutes. The resultant pellet was re-suspended into vaccine freezing medium (RPMI 1640/8%FCS/8%DMSO) at 5 x 10 6 cells per ml of freezing medium. Suspensions were then aliquoted into cryovials at 1.2ml per vial; vials were then placed into a -80°C freezer overnight prior to storage in liquid nitrogen.
  • vaccine freezing medium RPMI 1640/8%FCS/8%DMSO
  • the challenge material used for these experiments is the cell line Mat-Ly-Lu (wild type).
  • Mat-Ly-Lu cells(1 x 10 6 ) were seeded into a T175 tissue culture flask in 35 ml of RPMI 1640 culture medium supplemented with 10% FCS/2 mM Glutamine. Cells were allowed to grow until confluent under conditions of 37°C/5% CO 2 .
  • Confluent cells were harvested by washing the monolayer with 10ml Hank's buffered salt solution, followed by 5ml trypsin/EDTA solution to remove cells from the flask. Once all cells had lifted away from the flask surface the trypsin reaction was stopped by the addition of 5ml FCS. The 10ml of cell suspension was collected in a 50ml tube, centrifuged at 18,000 rpm for 3 minutes. The resultant pellet was re-suspended into 10ml PBS and stored on ice. A cell count was performed as detailed above and cell suspension adjusted to 5 x 10 5 per ml and aliquoted into cryovials at 1.2 ml per vial. Vials were then held on ice prior to injection.
  • Mat-Ly-Lu cells prepared as in the previous section were injected into shaved left hind flank of a copenhagen rat. 1 vial is sufficient to inject 5 animals (1 experimental group). See below for injection regime for protection and therapy
  • Tumor dimensions were collected using calipers, two measurements-longitudinal and transverse were taken for each tumor. These values were multiplied together to give an indication of tumor size. A kill size of 25 mm was set such that any tumor reaching this size in one of the two measurable dimension was culled by exposure to a rising concentration of CO2 and cervical dislocation. Kill curves were then generated.
  • Results for the comparison of 3D grown vaccines is shown in Figure 7.
  • the 3D grown PA3 cells offer a good level of protection above that of the vehicle control.
  • the 3D co-culture of PA3 with YPEN does not confer any protection and may reflect the reduction of PA3 cells as a mention previously .
  • PA3 cells with RBMS also offer a good level of protection even though the level of PA3 is reduced compared to the PA3 alone vaccine.
  • the co-culture of PA3/YPEN and RBMS provides slightly better protection than does the PA3 vaccine even though the PA3 contribution is probably three fold lower due to the seeeding level in the 3D culture. This would suggest that the co-culture of cell has resulted in an improved repertoire of antigens more reflective of the tumour in situ even though the vaccine is allogeneic.
  • 10608 3868 0.36463 syntaxin 7 12068 6016 0.498508 ras-related protein RAB-32 5600 112 0.02 alcohol dehydrogenase class II pi subunit 5780 644 0.111419 peroxisomal bifunctonal enzyme 18800 10588 0.563191 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase; HMGCR) 6548 828 0.126451 apolipoprotein D precursor (apo-D) 13652 8300 0.60797 dihydrofolate reductase (DHFR) 15652 10956 0.699974 homolog of yeast D2-isopentenylpyrophosphate isomerase (IPP isomerase) 10036 1888 0.188123 LOX (Protein-lysine 6-oxidase, Lysyl-Oxidase) 15136 10044 0.663584 TIA-1 related protein; nucleolysin TIAR 5648
  • microtubule-associated protein tau (MAPT); MTBT1 7540 1100 0.145889 PROTEIN PHPS1-2 7428 916 0.123317 epilepsy holoprosencephaly candidate-1 protein (EHOC-1); transmembrane protein 1 13184 6080 0.461165 DNA polymerase gamma (POLG); mitochondrial DNA polymerase Genes Up-Regulated in DU-145 / NHBSF Co-Culture Spot Intensity and Ratio DU-145/NHBSF Co-culture Ratio 3768 10280 2.728238 ESE1, ERT, JEN, ELF3 (Epithelial-specific transcription factor) 3732 13808 3.699893 Myelin-oligodendrocyte glycoprotein precursor (MOG) 19928 32924 1.652148 HLA-B-associated transcript 2; large proline-rich protein BAT2 2388 9900 4.145729 Complement 3 (C3) 6552 14756 2.252137 breakpoint

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Claims (30)

  1. Vaccin contre le cancer à base de cellules complètes ayant :
    un composant immunogène qui comprend des cellules malignes et non malignes co-cultivées; et
    un véhicule, un excipient ou un diluant pharmaceutiquement acceptables.
  2. Vaccin à base de cellules complètes selon la revendication 1, dans lequel les cellules co-cultivées ont une immunogénicité modifiée.
  3. Vaccin à base de cellules complètes selon la revendication 2, dans lequel les cellules co-cultivées ont été cultivées dans des conditions qui n'étaient pas des conditions de culture 2D statique conventionnelle et dans lequel l'immunogénicité des cellules co-cultivées a été modifiée en comparaison à l'immunogénicité de cellules équivalentes co-cultivées dans des conditions de culture 2D statique conventionnelle.
  4. Vaccin contre le cancer à base de cellules complètes selon la revendication 2 ou 3, dans lequel l'immunogénicité modifiée est causée par la présentation de peptides sur des molécules MHC-1 des cellules non malignes.
  5. Vaccin contre le cancer à base de cellules complètes selon la revendication 1, dans lequel les cellules co-cultivées sont sous forme d'un sphéroïde.
  6. Vaccin à base de cellules complètes selon la revendication 5, dans lequel une ou plusieurs des cellules co-cultivées du sphéroïde, a/ont une immunogénicité modifiée.
  7. Vaccin à base de cellules complètes selon la revendication 6, dans lequel les cellules co-cultivées ont été co-cultivées sous microgravité et la ou chaque cellule co-cultivée a une immunogénicité modifiée comparée à des cellules équivalentes co-cultivées dans des conditions de culture 2D statique conventionnelle.
  8. Vaccin contre le cancer à base de cellules complètes selon la revendication 6 ou 7, dans lequel l'immunogénicité modifiée est causée par la présentation de peptides sur des molécules MHC-1 des cellules non malignes.
  9. Vaccin contre le cancer à base de cellules complètes selon l'une quelconque des revendications précédentes, dans lequel le composant immunogène comprend en plus un adjuvant.
  10. Vaccin à base de cellules complètes selon l'une quelconque des revendications précédentes, dans lequel les cellules non malignes sont autologues et les cellules malignes sont allogéniques.
  11. Vaccin contre le cancer à base de cellules complètes selon l'une quelconque des revendications précédentes, dans lequel les cellules malignes et/ou non malignes sont dérivées d'une lignée cellulaire.
  12. Procédé pour produire un vaccin contre le cancer à base de cellules complètes qui comprend :
    co-cultiver des cellules malignes et non malignes;
    récolter les cellules co-cultivées;
    ajouter les cellules co-cultivées récoltées à un véhicule, excipient ou diluant pharmaceutiquement acceptables.
  13. Procédé selon la revendication 12, dans lequel les cellules sont co-cultivées dans des conditions qui ne sont pas des conditions de culture 2D statique conventionnelle.
  14. Procédé selon la revendication 13, dans lequel les cellules sont co-cultivées dans des conditions de culture non adhérentes.
  15. Procédé selon la revendication 14, dans lequel les cellules sont co-cultivées dans des récipients de culture non adhérents.
  16. Procédé selon la revendication 13, dans lequel les cellules sont co-cultivées dans des conditions de culture non statiques adhérentes.
  17. Procédé selon la revendication 16, dans lequel les cellules sont co-cultivées dans des flacons de culture roulants.
  18. Procédé selon la revendication 13, dans lequel les cellules sont co-cultivées sous microgravité.
  19. Procédé selon la revendication 18, dans lequel les cellules forment des sphéroïdes sous microgravité et les sphéroïdes sont récoltés et ajoutés au véhicule, excipient ou diluant pharmaceutiquement acceptables.
  20. Procédé selon la revendication 13, dans lequel les cellules sont co-cultivées sur des micro-supports mis en suspension dans un milieu de culture.
  21. Procédé selon la revendication 20, dans lequel les micro-supports sont des perles ou des disques en fibres non tissées.
  22. Procédé selon l'une quelconque des revendications 12 à 21, dans lequel co-cultiver les cellules modifie leur immunogénicité.
  23. Procédé selon la revendication 22, dans lequel l'immunogénicité des cellules est modifiée en comparaison à des cellules équivalentes co-cultivées dans des conditons de culture 2D statique conventionnelle.
  24. Procédé selon la revendication 22 ou 23, dans lequel l'immunogénicité modifiée est causée par la présentation de peptides sur des molécules MHC-1 des cellules non malignes.
  25. Procédé selon l'une quelconque des revendications 12 à 24, dans lequel les cellules non malignes sont autologues et les cellules malignes sont allogéniques.
  26. Procédé selon l'une quelconque des revendications 12 à 25, dans lequel les cellules malignes et/ou non malignes sont dérivées d'une lignée cellulaire.
  27. Procédé selon l'une quelconque des revendications 12 à 26 comprenant en plus l'irradiation gamma des cellules co-cultivées.
  28. Procédé selon l'une quelconque des revendications 12 à 27, dans lequel les cellules comprennent l'un ou les deux de ECACC 00032802 et de ECACC 00032801.
  29. Vaccin selon l'une quelconque des revendications 1 à 11, dans lequel les cellules comprennent l'un ou les deux de ECACC 00032802 et de ECACC 00032801.
  30. Vaccin selon l'une quelconque des revendications 1 à 11 et 29, dans lequel le cancer à traiter est un cancer de la prostate.
EP00931400A 1999-05-21 2000-05-19 Vaccin anticancereux a cellules entieres comprenant des cellules malignes et non-malignes co-cultivees Expired - Lifetime EP1178822B1 (fr)

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GBGB9911823.4A GB9911823D0 (en) 1999-05-21 1999-05-21 New vaccine formulations
GB9911824 1999-05-21
GB9911823 1999-05-21
GBGB9911824.2A GB9911824D0 (en) 1999-05-21 1999-05-21 New vaccine formulations
GB0008032 2000-04-01
GB0008029 2000-04-01
GB0008029A GB0008029D0 (en) 2000-04-01 2000-04-01 New vaccine formulations
GB0008032A GB0008032D0 (en) 2000-04-01 2000-04-01 New prostate cell lines
PCT/GB2000/001918 WO2000071155A2 (fr) 1999-05-21 2000-05-19 Nouvelles formulations vaccinales -2

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